Cd117 pe cy7

When the target cells were cell lines or T cells, incubation of the cells for 30 min was done with CD44v6‐PE (BD Bioscience, USA) or isotype control mouse IgG1‐PE (BD Bioscience, USA). When the target cells were peripheral blood mononuclear cells (PBMCs) from AML patients or healthy donors, the PBMCs were incubated with the following antibodies (BD Bioscience, USA): Fixable Viability Stain 510, CD117‐PE‐CY7, CD34‐APC, CD45‐APC‐CY7, HLA‐DR‐BV421, CD44v6‐PE or mouse IgG1‐PE. The samples were all detected by BD LSRFortessa X‐20 flow cytometry and analysed by FlowJo software.

Primary splenocytes and thymocytes were recovered by crushing excised spleen or thymic tissue between glass microscope slides, washing with PBS + 2% FBS, and passing through an 80 µm mesh filter (Sefar, Nitex 03-80/37). Cells were washed once in PBS + 2% FBS and were resuspended in PBS + 2% FBS and kept on ice until analysis. For analysis of bone marrow, marrow was extracted from femurs using a mortar and pestle and processed as above. For analysis of peripheral blood, red blood cells were lysed (155 mM NH₄CL, 12 mM NaHCO₃, 0.13 mM EDTA) at room temperature and washed twice in PBS + 2% FBS. Flow cytometry analysis included a 4-color T-cell panel (CD8a-PE, CD4-APC, CD19-APC-Cy7and CD45r/B220-PerCP (BD Biosciences)) and a 6-color erythroid/myeloid panel (CD45-APC-Cy7, CD71-FITC, CD117-PE-Cy7, Ter119-APC, Ly-6G/Ly-6C (Gr1)-PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend). Cells were also stained with DAPI to determine cell viability. Flow cytometry was performed on a Canto 2 (Becton Dickinson) or a custom designed LSR2 (Becton Dickinsin, Franklin Lakes, NJ) and analyzed using either FlowJo Software (FlowJo, Ashland, OR) or Woodlist, a noncommercial software program developed in our clinical laboratory^{52 (link)}.